DESCRIPTION: Post-translational modifications (PTMs) of histones play a crucial role in the dynamic regulation of the physiological state of our genome. Regulatory proteins dock onto PTMs for anchorage on our genome to perform site-specific regulatory functions. PTMs also disrupt the anchorage of proteins that specifically bind PTM-less (unmodified) histones. However, much less is known about proteins that bind PTM-less histones even though mutations in them cause inherited disorders. In addition, the current knowledge about the enzymes that introduce/remove histone PTMs is obtained using cell-free or fixed cell contexts providing poor information on the dynamic nature of histone PTMs. Taken together, these significantly contribute to the lack of current understanding of our genome regulation. Continued existence of this knowledge gap represents an important problem because, until it is filled, understanding the initiation, progression of various diseases due to histone PTM aberrations remain incomprehensible. Our long-term goal is to better understand the role of PTMs in genome regulation. The objective for this particular R15 application is to identify putative human proteins capable of interacting with PTM-less histones to investigate their role in various disorders, and to design sensitive enzyme sensors for reporting enzymatic activity in living cells. The central hypothesis is that in the family of protein modules that interact with PTMs, there are members capable of interacting with PTM-less peptide and they possess specific sequence patterns. This is formulated on strong preliminary data from the applicant's laboratory. Capturing such interactions in crude cellular extracts permits rapid screening of a sensitive sensor among futile alternatives. Our rationale is that experimentally verified large-scale identification of proteins that tether onto PTM-less histones will provide new insights on genome regulation, enabling studies on the interplay between global histone mark maps and the localization of regulatory complexes. The availability of sensitive enzyme sensors will enable comparison of the action of cellular programming machinery between living normal and diseased cells. The two specific aims are: 1) Identify unmodified Arg/Lys readers in the family known to recognize methylated-Lys/Arg; and 2) Design sensitive sensors for histone H3- Lys4 methylases. In Aim-1, we test PTM-less histone binding capability of 8 putative human proteins, effect of point mutations in such interactions and examine the effect of PTMs on such interaction. Using rapid binding assays we infer about these interactions. For Aim-2, we search sensor ON-state with the highest reporting efficiency among a library of alternatives. The approach is innovative for the rapidity in the searches. The research is significant, because it is expected to vertically expand the understanding of histone PTMs in genome regulation. That will enable preventative, therapeutic manipulations of histone PTM aberrations in diseases.